Special article

The local regulation of cerebral blood flow☆

With the aim to obtain potent adenosine A_2A receptor (A_2AR) ligands, a series of eighteen derivatives of 4-hydroxy-N-(4-methoxy-7-morpholin-4-yl-1,3-benzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide (SYN-115, Tozadenant) were designed and synthesized. The target compounds were obtained by a chemical building block principle that involved reaction of the appropriate aminobenzothiazole phenyl carbamates with either commercially available or readily synthesized functionalized piperidines. Their affinity and subtype selectivity with regard to human adenosine A₁-and A_2A receptors were determined using radioligand binding assays. K_i values for human A_2AR ranged from 2.4 to 38 nM, with more than 120-fold selectivity over A₁ receptors for all evaluated compounds except 13k which had a K_i of 361 nM and 18-fold selectivity. The most potent fluorine-containing derivatives 13e, 13g and 13l exhibited K_i values of 4.9 nM, 3.6 nM and 2.8 nM for the human A_2AR. Interestingly, the corresponding values for rat A_2AR were found to be four to five times higher. Their binding to A_2AR was further confirmed by radiolabeling with ¹⁸F and in vitro autoradiography in rat brain slices, which showed almost exclusive striatal binding and complete displacement by the A_2AR antagonist ZM 241385. We conclude that these compounds represent potential candidates for the visualization of the A_2A receptor and open pathways to novel therapeutic treatments of neurodegenerative disorders or cancer.

This chapter discusses the various receptor mediated events in the microcirculation focusing on the various kinds of receptors. Epinephrine (Epi) and norepinephrine (NE) are the main endogenous catecholamines mediating sympathetic regulation of the microcirculation. Both vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) express multiple adrenoceptor subtypes. All three α₁-adrenoceptor subtypes contribute to vasoconstriction in the microcirculation. The diverse family of adrenoceptors mediates multiple important but opposing effects in the microcirculation. Dopamine receptors are grouped into the D₁ and D₂ families. Dopamine is thought to bind to five subtypes of receptors. Microcirculatory effects of the D₁ and D₂ receptor families have been reported, with primary focus on the D₁ family. -hydroxytryptamine (5-HT, serotonin) has a rich history in the vasculature. The detection of M₁, M₃, and possibly M₅ muscarinic receptor subtypes in brain microcirculation is consistent with reports showing that these receptors mediate vasoconstriction, vasodilatation, and activation of nitric oxide synthase. ANG II receptors have a variety of important influences in the control of the vasculature and especially the microcirculation. ANG II has multiple actions in the vasculature, including regulation of vascular tone, microvessel density (MVD) and vascular structure. Arginine vasopressin receptors, endothelin receptors, adrenomedullin/cgrp receptors, adenosine receptors, purinergic receptors, kinin receptors, histamine receptors, eicosanoid receptors, and growth factor receptors are also explored.

Adenosine is a recognized inhibitory neuromodulator and neuroprotective agent in the central nervous system. It is produced both intra- and extracellularly and transported across the cell membrane. Nucleoside transporters thus have a major impact on the extracellular adenosine levels, and consequently adenosine signalling.

We have raised and characterized polyclonal antibodies against both the equilibrative nucleoside transporters 1 and 2, and report for the first time their distribution in rat brain at the cellular level. Double staining studies were performed to assess the localization of the transporters in neural and glial cells.

Both transporters were present in practically all neurons. Some astrocytes showed equilibrative nucleoside transporter 1 staining, while equilibrative nucleoside transporter 2 staining on astrocytes was observed only sporadically.

The important roles played by the A₁ adenosine receptor (A₁AR) in brain physiology and pathology make this receptor a target for in vivo imaging. Here we describe the distribution of A₁ARs in the living human brain with PET, made possible for the first time by the highly potent and selective A₁AR antagonist 8-cyclopentyl-3-(3-[¹⁸F]fluoropropyl)-1-propylxanthine ([¹⁸F]CPFPX). In vivo data demonstrate a rapid cerebral uptake, peaking at 2.9 ± 0.6% injected dose/liter at 3.3 ± 1.3 min, followed by a gradual washout. Consistent with the results of autoradiography, high receptor densities occurred in the putamen and the mediodorsal thalamus. Neocortical regions showed regional differences in [¹⁸F]CPFPX binding, with high accumulation in temporal > occipital > parietal > frontal lobes and a lower level of binding in the sensorimotor cortex. Ligand accumulation was low in cerebellum, midbrain, and brain stem. Metabolism of [¹⁸F]CPFPX is rapid outside the central nervous system, but the metabolites do not penetrate the blood–brain barrier. In conclusion, in vivo application of [¹⁸F]CPFPX, a highly potent and selective PET ligand, for the first time allows the imaging of A₁ARs in the living human brain.

Adenosine exerts two parallel modulatory roles in the CNS, acting as a homeostatic modulator and also as a neuromodulator at the synaptic level. We will present evidence to suggest that these two different modulatory roles are fulfilled by extracellular adenosine originated from different metabolic sources, and involve receptors with different sub-cellular localisation. It is widely accepted that adenosine is an inhibitory modulator in the CNS, a notion that stems from the preponderant role of inhibitory adenosine A₁ receptors in defining the homeostatic modulatory role of adenosine. However, we will review recent data that suggests that the synaptically localised neuromodulatory role of adenosine depend on a balanced activation of inhibitory A₁ receptors and mostly facilitatory A_2A receptors. This balanced activation of A₁ and A_2A adenosine receptors depends not only on the transient levels of extracellular adenosine, but also on the direct interaction between A₁ and A_2A receptors, which control each other’s action.

This study aimed to evaluate the role for adenosine A_2A receptors in the autoregulatory vasodilation to hypotension in relation with cerebral blood flow (CBF) autoregulation in rat pial arteries. Changes in pial artery diameters were observed directly through a closed cranial window. Vasodilation induced by adenosine was markedly suppressed by ZM 241385 (1 μmol/l, A_2A antagonist) and alloxazine (1 μmol/l, A_2B antagonist), but not by 8-cyclopentyltheophylline (CPT, 1 μmol/l, A₁ antagonist). CGS-21680-induced vasodilation was more strongly inhibited by ZM 241385 (25.3-fold; P<0.05) than by alloxazine. In contrast, 5′-N-ethylcarboxamido-adenosine (NECA)-induced vasodilation was more prominently suppressed by alloxazine (12.0-fold; P<0.001) than by ZM 241385. The autoregulatory vasodilation in response to acute hypotension of the pial arteries was significantly suppressed by ZM 241385, but not by CPT and alloxazine. Consistent with this finding, the lower limit of CBF autoregulation significantly shifted to a higher blood pressure by 1 μmol/l of ZM 241385 (53.0±3.9 mmHg to 69.2±2.9 mmHg, P<0.01) and 10 μmol/l of glibenclamide (54.7±6.5 mmHg to 77.9±4.2 mmHg, P<0.001), but not by CPT and alloxazine. Thus, it is suggested that adenosine-induced vasodilation of the rat pial artery is mediated via activation of adenosine A_2A and A_2B receptors, but not by A₁ subtype, and activation of adenosine A_2A receptor preferentially contributes to the autoregulatory vasodilation via activation of ATP-sensitive K⁺ channels in response to hypotension and maintenance of CBF autoregulation.